Methods for the identification of inhibitors of 5-aminolevulinate synthase as antibiotics

ABSTRACT

The present inventors have discovered that 5-Aminolevulinate synthase is essential for fungal pathogenicity. Specifically, the inhibition of 5-Aminolevulinate synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, 5-Aminolevulinate synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit 5-Aminolevulinate synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

[0001] The invention relates generally to methods for the identificationof antibiotics, preferably antifuingals that affect the biosynthesis ofheme.

BACKGROUND OF THE INVENTION

[0002] Filamentous fungi are the causal agents responsible for manyserious pathogenic infections of plants and animals. Since fungi areeukaryotes, and thus more similar to their host organisms than, forexample bacteria, the treatment of infections by fungi poses specialrisks and challenges not encountered with other types of infections. Onesuch fungus is Magnaporthe grisea, the fungus that causes rice blastdisease. It is an organism that poses a significant threat to foodsupplies worldwide. Other examples of plant pathogens of economicimportance include the pathogens in the genera Agaricus, Alternaria,Anisogramma, Anthracoidea, Antrodia, Apiognomonia, Apiosporina,Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera,Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora,Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria,Chrysomyxa, Cladosporium, Claviceps, Cochliobolus, Coleosporium,Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium,Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora,Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia,Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,Endocronartium, Endothia, Entyloma, Epichloe, Erysiphe, Exobasidium,Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma,Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella,Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium,Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus,Irpex, Kabatiella, Kabatina, Laetiporus, Laetisaria, Lasiodiplodia,Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina,Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina,Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium,Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella,Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma,Penicillium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus,Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis,Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera,Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium,Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina,Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia,Typhula, Uncinula, Urocystis, Uromyces, Ustilago, Valsa, Venturia,Verticillium, Xylaria, and others. Related organisms in theclassification, oomycetes, that include the genera Albugo, Aphanomyces,Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara,Pseudoperonospora, Pythium, Sclerophthora, and others are alsosignificant plant pathogens and are sometimes classified along with thetrue fungi. Human diseases that are caused by filamentous fungi includelife-threatening lung and disseminated diseases, often a result ofinfections by Aspergillus fumigatus. Other fungal diseases in animalsare caused by fungi in the genera, Fusarium, Blastomyces, Microsporum,Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,Cryptococcus, other Aspergilli, and others. The control of fungaldiseases in plants and animals is usually mediated by chemicals thatinhibit the growth, proliferation, and/or pathogenicity of the fungalorganisms. To date, there are less than twenty known modes-of-action forplant protection fungicides and human antifungal compounds.

[0003] A pathogenic organism has been defined as an organism thatcauses, or is capable of causing disease. Pathogenic organisms propagateon or in tissues and may obtain nutrients and other essential materialsfrom their hosts. A substantial amount of work concerning filamentousfungal pathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al. (1999) MicrobPathog 27: 123-31 (PMID: 10455003)) have shown that the deletion ofeither of two suspected pathogenicity related genes encoding an alkalineprotease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encoding,aspergillopepsin (PEP) in Aspergillus fumigatus, had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) U.S. Pat. Nos.4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co.Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) MolMicrobiol 36: 1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

[0004] There are also a number of papers that report less clear results,showing neither full pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the area transcriptional activator on the pathogenicity ofAspergillus fumigatus.

[0005] Therefore, it is not currently possible to determine whichspecific growth materials may be readily obtained by a pathogen from itshost, and which materials may not. We have found that Magnaporthe griseathat cannot synthesize their own heme are non-pathogenic on their hostorganism. In addition to being a key component of respiratorycytochromes and hemoglobin, heme is the prosthetic group for manyenzymes involved in the detoxification of oxygen radicals and in themetabolism of fatty acids and sterols. In yeast, Saccharomycescerevisiae, mutants deficient in heme biosynthesis have been isolatedand genetically studied in detail (Gollub et al. (1977) J Biol Chem 252:2846-54 (PMID: 323256)). The 5-aminolevulinate synthase gene has beencloned from Aspergillus oryzae and shown to be used as a selectablemarker for the transformation of A. oryzae (Elrod et al. (2000) CurrGenet 38: 291-8 (PMID: 11191214)). In humans, two 5-aminolevulinatesynthase genes have been identified. Mutations in one of them, encodingan erythroid isoform, result in X-linked sideroblastic anemia (Cox etal. (1994) N Engl J Med 330: 675-9 (PMID: 8107717)). 5-aminolevulinatesynthase has been proposed as a new antimalarial target (Padmanaban andRangarajan (2000) Biochem Biophys Res Commun 268: 665-8 (PMID:10679261)).

[0006] To date there do not appear to be any publications demonstratingan anti-pathogenic effect of the knock-out, over-expression, antisenseexpression, or inhibition of the genes or gene products involved in hemebiosynthesis in filamentous fungi. Thus, it has not been shown that thede novo biosynthesis of heme is essential for fungal pathogenicity. And,thus, it would be desirable to determine the utility of the enzymesinvolved in heme biosynthesis for evaluating antibiotic compounds,especially fungicides. If a fungal biochemical pathway or specific geneproduct in that pathway is shown to be required for fungalpathogenicity, various formats of in vitro and in vivo screening assaysmay be put in place to discover classes of chemical compounds that reactwith the validated target gene, gene product, or biochemical pathway,and are thus candidates for antifungal, biocide, and biostaticmaterials.

SUMMARY OF THE INVENTION

[0007] Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding 5-Aminolevulinate synthase inMagnaporthe grisea prevents or inhibits the pathogenicity of the fungus.Thus, the present inventors have discovered that 5-Aminolevulinatesynthase is essential for normal rice blast pathogenicity, and can beused as a target for the identification of antibiotics, preferablyfungicides. Accordingly, the present invention provides methods for theidentification of compounds that inhibit 5-Aminolevulinate synthaseexpression or activity. The methods of the invention are useful for theidentification of antibiotics, preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 shows the reaction performed by 5-Aminolevulinate synthase(ALAS1) reaction. The Substrates/Products are succinyl-CoA and glycineand the Products/Substrates are 5-aminolevulinate, CoA, and CO₂. Thefunction of the 5-Aminolevulinate synthase enzyme is the interconversionof succinyl-CoA and glycine to 5-aminolevulinate, CoA, and CO₂. Thisreaction is part of the heme biosynthesis pathway.

[0009]FIG. 2 shows a digital image showing the effect of ALAS1 genedisruption on Magnaporthe grisea pathogenicity using whole plantinfection assays. Rice variety CO39 was inoculated with wild-type andthe transposon insertion strains, KO1-1 and KO1-106. Leaf segments wereimaged at five days post-inoculation.

[0010]FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-1 andKO1-106, were grown in (A) minimal media and (B) minimal media with theaddition of 5-aminolevulinate, respectively. The x-axis shows time indays and the y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (—♦—), transposon strainKO1-1 (—▪—), and transposon strain KO1-106 (—▴—).

DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise indicated, the following terms are intended tohave the following meanings in interpreting the present invention.

[0012] The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

[0013] As used herein, the term “ALAS1” means a gene encoding5-Aminolevulinate synthase activity, referring to an enzyme thatcatalyses the interconversion of succinyl-CoA and glycine with5-aminolevulinate, CoA, and CO₂, and may also be used to refer to thegene product.

[0014] As used herein, the term “allele” refers to any of thealternative forms of a gene that may occur at a given locus.

[0015] As used herein, the terms “5-Aminolevulinate synthase” (EC2.3.1.37) and “5-Aminolevulinate synthase polypeptide” are synonymouswith “the ALAS1 gene product” and refer to an enzyme that catalyses theinterconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA,and CO₂.

[0016] The term “antibiotic” refers to any substance or compound thatwhen contacted with a living cell, organism, virus, or other entitycapable of replication, results in a reduction of growth, viability, orpathogenicity of that entity.

[0017] The term “binding” refers to a non-covalent or a covalentinteraction, preferably non-covalent, that holds two molecules together.For example, two such molecules could be an enzyme and an inhibitor ofthat enzyme. Non-covalent interactions include hydrogen bonding, ionicinteractions among charged groups, van der Waals interactions andhydrophobic interactions among nonpolar groups. One or more of theseinteractions can mediate the binding of two molecules to each other.

[0018] The term “biochemical pathway” or “pathway” refers to a connectedseries of biochemical reactions normally occurring in a cell, or morebroadly a cellular event such as cellular division or DNA replication.Typically, the steps in such a biochemical pathway act in a coordinatedfashion to produce a specific product or products or to produce someother particular biochemical action. Such a biochemical pathway requiresthe expression product of a gene if the absence of that expressionproduct either directly or indirectly prevents the completion of one ormore steps in that pathway, thereby preventing or significantly reducingthe production of one or more normal products or effects of thatpathway. Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

[0019] As used herein, the term “cDNA” means complementarydeoxyribonucleic acid.

[0020] As used herein, the term “CoA” means coenzyme A.

[0021] As used herein, the term “conditional lethal” refers to amutation permitting growth and/or survival only under special growth orenvironmental conditions.

[0022] As used herein, the term “cosmid” refers to a hybrid vector, usedin gene cloning, that includes a cos site (from the lambdabacteriophage). It also contains drug resistance marker genes and otherplasmid genes. Cosmids are especially suitable for cloning large genesor multigene fragments.

[0023] As used herein, the term “dominant allele” refers to a dominantmutant allele in which a discernable mutant phenotype can be detectedwhen this mutation is present in an organism that also contains a wildtype (non-mutant), recessive allele, or other dominant allele.

[0024] As used herein, the term “DNA” means deoxyribonucleic acid.

[0025] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay.

[0026] “Fungi” (singular: fungus) refers to whole fungi, fungal organsand tissues (e.g., asci, hyphae, pseudohyphae, rhizoid, sclerotia,sterigmata, spores, sporodochia, sporangia, synnemata, conidia,ascostroma, cleistothecia, mycelia, perithecia, basidia and the like),spores, fungal cells and the progeny thereof. Fungi are a group oforganisms (about 50,000 known species), including, but not limited to,mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi.They can either exist as single cells or make up a multicellular bodycalled a mycelium, which consists of filaments known as hyphae. Mostfungal cells are multinucleate and have cell walls, composed chiefly ofchitin. Fungi exist primarily in damp situations on land and, because ofthe absence of chlorophyll and thus the inability to manufacture theirown food by photosynthesis, are either parasites on other organisms orsaprotrophs feeding on dead organic matter. The principal criteria usedin classification are the nature of the spores produced and the presenceor absence of cross walls within the hyphae. Fungi are distributedworldwide in terrestrial, freshwater, and marine habitats. Some live inthe soil. Many pathogenic fungi cause disease in animals and man or inplants, while some saprotrophs are destructive to timber, textiles, andother materials. Some fungi form associations with other organisms, mostnotably with algae to form lichens.

[0027] As used herein, the term “fungicide”, “antifungal”, or“antimycotic” refers to an antibiotic substance or compound that killsor suppresses the growth, viability, or pathogenicity of at least onefungus, fungal cell, fungal tissue or spore.

[0028] In the context of this disclosure, “gene” should be understood torefer to a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides.) The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

[0029] As used in this disclosure, the terms “growth” or “cell growth”of an organism refers to an increase in mass, density, or number ofcells of said organism. Some common methods for the measurement ofgrowth include the determination of the optical density of a cellsuspension, the counting of the number of cells in a fixed volume, thecounting of the number of cells by measurement of cell division, themeasurement of cellular mass or cellular volume, and the like.

[0030] As used in this disclosure, the term “growth conditionalphenotype” indicates that a fungal strain having such a phenotypeexhibits a significantly greater difference in growth rates in responseto a change in one or more of the culture parameters than an otherwisesimilar strain not having a growth conditional phenotype. Typically, agrowth conditional phenotype is described with respect to a singlegrowth culture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

[0031] As used herein, the term “H₂O” means water.

[0032] As used herein, the term “heterologous ALAS1 gene” means a gene,not derived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea 5-Aminolevulinate synthase, preferably 25%, 50%, 75%,90%, 95%, 99% and each integer unit of activity from 10-100% inascending order.

[0033] As used herein, the term “His-Tag” refers to an encodedpolypeptide consisting of multiple consecutive histidine amino acids.

[0034] As used herein, the term “HPLC” means high pressure liquidchromatography.

[0035] As used herein, the terms “hph”, “hygromycin Bphosphotransferase”, and “hygromycin resistance gene” refer to the E.coli hygromycin phosphotransferase gene or gene product.

[0036] As used herein, the term “hygromycin B” refers to anaminoglycosidic antibiotic, used for selection and maintenance ofeukaryotic cells containing the E. coli hygromycin resistance gene.

[0037] “Hypersensitive” refers to a phenotype in which cells are moresensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0038] “Hyposensitive” refers to a phenotype in which cells are lesssensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0039] As used herein, the term “imperfect state” refers to aclassification of a fungal organism having no demonstrable sexual lifestage.

[0040] The term “inhibitor”, as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity of 5-Aminolevulinatesynthase or substantially reduces the level of enzymatic activity,wherein “substantially” means a reduction at least as great as thestandard deviation for a measurement, preferably a reduction by 50%,more preferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0041] A polynucleotide may be “introduced” into a fungal cell by anymeans known to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

[0042] As used herein, the term “knockout” or “gene disruption” refersto the creation of organisms carrying a null mutation (a mutation inwhich there is no active gene product), a partial null mutation ormutations, or an alteration or alterations in gene regulation byinterrupting a DNA sequence through insertion of a foreign piece of DNA.Usually the foreign DNA encodes a selectable marker.

[0043] As used herein, the term “LB agar” means Luria's Broth agar.

[0044] The term “method of screening” means that the method is suitable,and is typically used, for testing for a particular property or effectin a large number of compounds. Typically, more than one compound istested simultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “Method ofscreening” also refers to the determination of a set of differentproperties or effects of one compound simultaneously.

[0045] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0046] As used herein, the term “mutant form” of a gene refers to a genewhich has been altered, either naturally or artificially, changing thebase sequence of the gene. The change in the base sequence may be ofseveral different types, including changes of one or more bases fordifferent bases, deletions, and/or insertions, such as by a transposon.By contrast, a normal form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

[0047] As used herein, the term “Ni” refers to nickel.

[0048] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0049] As used herein, a “normal” form of a gene (wild type) is a formcommonly found in natural populations of an organism. Commonly a singleform of a gene will predominate in natural populations. In general, sucha gene is suitable as a normal form of a gene, however, other formswhich provide similar functional characteristics may also be used as anormal gene. In particular, a normal form of a gene does not confer agrowth conditional phenotype on the strain having that gene, while amutant form of a gene suitable for use in these methods does providesuch a growth conditional phenotype.

[0050] As used herein, the term “one form” of a gene is synonymous withthe term “gene”, and a “different form” of a gene refers to a gene thathas greater than 49% sequence identity and less than 100% sequenceidentity with said first form.

[0051] As used herein, the term “pathogenicity” refers to a capabilityof causing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

[0052] As used herein, the term “PCR” means polymerase chain reaction.

[0053] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W.Gish, et al (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at theNational Center for Biotechnology or using Smith Waterman Alignment(Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195-7 (PMID:7265238)) as incorporated into GeneMatcher Plus™. It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

[0054] By “polypeptide” is meant a chain of at least two amino acidsjoined by peptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

[0055] As used herein, the term “proliferation” is synonymous to theterm “growth”.

[0056] As used herein, the term “reverse transcriptase-PCR” meansreverse transcription-polyrnerase chain reaction.

[0057] As used herein, the term “RNA” means ribonucleic acid.

[0058] As used herein, “semi-permissive conditions” are conditions inwhich the relevant culture parameter for a particular growth conditionalphenotype is intermediate between permissive conditions andnon-permissive conditions. Consequently, in semi-permissive conditionsan organism having a growth conditional phenotype will exhibit growthrates intermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially finctionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

[0059] “Sensitivity phenotype” refers to a phenotype that exhibitseither hypersensitivity or hyposensitivity.

[0060] The term “specific binding” refers to an interaction between5-Aminolevulinate synthase and a molecule or compound, wherein theinteraction is dependent upon the primary amino acid sequence and/or theconformation of 5-Aminolevulinate synthase.

[0061] As used herein, the term “TLC” means thin layer chromatography.

[0062] “Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

[0063] For the purposes of the invention, “transgenic” refers to anycell, spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

[0064] As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/US00/07317, and U.S. Ser. No. 09/658859.

[0065] As used herein, the term “transposition” refers to a complexgenetic rearrangement process involving the movement or copying of apolynucleotide (transposon) from one location and insertion intoanother, often within or between a genome or genomes, or DNA constructssuch as plasmids, bacmids, and cosmids.

[0066] As used herein, the term “transposon” (also known as a“transposable element”, “transposable genetic element”, “mobileelement”, or “jumping gene”) refers to a mobile DNA element such asthose, for example, described in WO 00/55346, PCT/US00/07317, and U.S.Ser. No. 09/658859. Transposons can disrupt gene expression or causedeletions and inversions, and hence affect both the genotype andphenotype of the organisms concerned. The mobility of transposableelements has long been used in genetic manipulation, to introduce genesor other information into the genome of certain model systems.

[0067] As used herein, the term “Tween 20” means sorbitanmono-9-octadecenoate poly(oxy-1,1-ethanediyl).

[0068] As used in this disclosure, the term “viability” of an organismrefers to the ability of an organism to demonstrate growth underconditions appropriate for said organism, or to demonstrate an activecellular function. Some examples of active cellular functions includerespiration as measured by gas evolution, secretion of proteins and/orother compounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

[0069] The present inventors have discovered that disruption of theALAS1 gene and/or gene product inhibits the pathogenicity of Magnaporthegrisea. Thus, the inventors are the first to demonstrate that5-Aminolevulinate synthase is a target for antibiotics, preferablyantifungals.

[0070] Accordingly, the invention provides methods for identifyingcompounds that inhibit ALAS1 gene expression or biological activity ofits gene product(s). Such methods include ligand binding assays, assaysfor enzyme activity, cell-based assays, and assays for ALAS1 geneexpression. Any compound that is a ligand for 5-Aminolevulinate synthasemay have antibiotic activity. For the purposes of the invention,“ligand” refers to a molecule that will bind to a site on a polypeptide.The compounds identified by the methods of the invention are useful asantibiotics.

[0071] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0072] a) contacting a 5-Aminolevulinate synthase polypeptide with atest compound; and

[0073] b) detecting the presence or absence of binding between said testcompound and said 5-Aminolevulinate synthase polypeptide;

[0074] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0075] The 5-Aminolevulinate synthase protein may have the amino acidsequence of a naturally occurring 5-Aminolevulinate synthase found in afungus, animal, plant, or microorganism, or may have an amino acidsequence derived from a naturally occurring sequence. Preferably the5-Aminolevulinate synthase is a fungal 5-Aminolevulinate synthase. ThecDNA (SEQ ID NO: 1) encoding the M. grisea 5-Aminolevulinate synthaseprotein, the genomic DNA (SEQ ID NO: 2) encoding the protein, and thepolypeptide (SEQ ID NO: 3) can be found herein.

[0076] In one aspect, the invention also provides for a polypeptideconsisting essentially of SEQ ID NO: 3. For the purposes of theinvention, a polypeptide consisting essentially of SEQ ID NO: 3 has atleast 80% sequence identity with SEQ ID NO: 3 and catalyses theinterconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA,and CO₂ with at least 10% of the activity of SEQ ID NO: 3. Preferably,the polypeptide consisting essentially of SEQ ID NO: 3 has at least 85%sequence identity with SEQ ID NO: 3, more preferably the sequenceidentity is at least 90%, most preferably the sequence identity is atleast 95% or 97 or 99%, or any integer from 80-100% sequence identity inascending order. And, preferably, the polypeptide consisting essentiallyof SEQ ID NO: 3 has at least 25%, at least 50%, at least 75% or at least90% of the activity of M. grisea 5-Aminolevulinate synthase, or anyinteger from 60-100% activity in ascending order.

[0077] By “fungal 5-Aminolevulinate synthase” is meant an enzyme thatcan be found in at least one fungus, and which catalyzes theinterconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA,and CO₂. The 5-Aminolevulinate synthase may be from any of the fungi,including ascomycota, zygomycota, basidiomycota, chytridiomycota, andlichens.

[0078] In one embodiment, the 5-Aminolevulinate synthase is aMagnaporthe 5-Aminolevulinate synthase. Magnaporthe species include, butare not limited to, Magnaporthe rhizophila, Magnaporthe salvinii,Magnaporthe grisea and Magnaporthe poae and the imperfect states ofMagnaporthe in the genus Pyricularia. Preferably, the Magnaporthe5-Aminolevulinate synthase is from Magnaporthe grisea.

[0079] In various embodiments, the 5-Aminolevulinate synthase can befrom Powdery Scab (Spongospora subterranea), Grey Mould (Botrytiscinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganodermaadspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilagomaydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercosporazeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillarialuteobubalina), Shoestring Rot (Armillaria ostoyae), Banana AnthracnoseFungus (Colletotrichum musae), Apple-rotting Fungus (Moniliniafructigena), Apple-rotting Fungus (Penicillium expansum), ClubrootDisease (Plasmodiophora brassicae), Potato Blight (Phytophthorainfestans), Root pathogen (Heterobasidion annosum), Take-all Fungus(Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), BeanRust (Uromyces appendiculatus), Northern Leaf Spot (Cochlioboluscarbonum), Milo Disease (Periconia circinata), Southern Corn Blight(Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), BrownStripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum),Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

[0080] Fragments of a 5-Aminolevulinate synthase polypeptide may be usedin the methods of the invention, preferably if the fragments include anintact or nearly intact epitope that occurs on the biologically activewildtype 5-Aminolevulinate synthase. The fragments comprise at least 10consecutive amino acids of a 5-Aminolevulinate synthase. Preferably, thefragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110,120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, or at least 610 consecutiveamino acids residues of a 5-Aminolevulinate synthase. In one embodiment,the fragment is from a Magnaporthe 5-Aminolevulinate synthase.Preferably, the fragment contains an amino acid sequence conserved amongfungal 5-Aminolevulinate synthases.

[0081] Polypeptides having at least 50% sequence identity with a fungal5-Aminolevulinate synthase are also useful in the methods of theinvention. Preferably, the sequence identity is at least 60%, morepreferably the sequence identity is at least 70%, most preferably thesequence identity is at least 80% or 90 or 95 or 99%, or any integerfrom 60-100% sequence identity in ascending order.

[0082] In addition, it is preferred that the polypeptide has at least10% of the activity of a fungal 5-Aminolevulinate synthase. Morepreferably, the polypeptide has at least 25%, at least 50%, at least 75%or at least 90% of the activity of a fungal 5-Aminolevulinate synthase.Most preferably, the polypeptide has at least 10%, at least 25%, atleast 50%, at least 75% or at least 90% of the activity of the M. grisea5-Aminolevulinate synthase protein.

[0083] Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

[0084] a) contacting a test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal 5-Aminolevulinate synthase; apolypeptide having at least 50% sequence identity with a fungal5-Aminolevulinate synthase; and a polypeptide having at least 10% of theactivity of a fungal 5-Aminolevulinate synthase; and

[0085] b) detecting the presence and/or absence of binding between saidtest compound and said polypeptide;

[0086] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0087] Any technique for detecting the binding of a ligand to its targetmay be used in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a 5-Aminolevulinate synthase protein or a fragment or variantthereof, the unbound protein is removed and the bound 5-Aminolevulinatesynthase is detected. In a preferred embodiment, bound 5-Aminolevulinatesynthase is detected using a labeled binding partner, such as a labeledantibody. In a variation of this assay, 5-Aminolevulinate synthase islabeled prior to contacting the immobilized candidate ligands. Preferredlabels include fluorescent or radioactive moieties. Preferred detectionmethods include fluorescence correlation spectroscopy (FCS) andFCS-related confocal nanofluorimetric methods.

[0088] Once a compound is identified as a candidate for an antibiotic,it can be tested for the ability to inhibit 5-Aminolevulinate synthaseenzymatic activity. The compounds can be tested using either in vitro orcell based assays. Alternatively, a compound can be tested by applyingit directly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

[0089] By decrease in growth, is meant that the antifungal candidatecauses at least a 10% decrease in the growth of the fungus or fungalcells, as compared to the growth of the fungus or fungal cells in theabsence of the antifungal candidate. By a decrease in viability is meantthat at least 20% of the fungal cells, or portion of the funguscontacted with the antifungal candidate are nonviable. Preferably, thegrowth or viability will be decreased by at least 40%. More preferably,the growth or viability will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal growth and cellviability are known to those skilled in the art. By decrease inpathogenicity, is meant that the antifungal candidate causes at least a10% decrease in the disease caused by contact of the fungal pathogenwith its host, as compared to the disease caused in the absence of theantifungal candidate. Preferably, the disease will be decreased by atleast 40%. More preferably, the disease will be decreased by at least50%, 75% or at least 90% or more. Methods for measuring fungal diseaseare well known to those skilled in the art, and include such metrics aslesion formation, lesion size, sporulation, respiratory failure, and/ordeath.

[0090] The ability of a compound to inhibit 5-Aminolevulinate synthaseactivity can be detected using in vitro enzymatic assays in which thedisappearance of a substrate or the appearance of a product is directlyor indirectly detected. 5-Aminolevulinate synthase catalyzes theirreversible or reversible reaction succinyl-CoA andglycine=5-aminolevulinate, CoA, and CO₂ (see FIG. 1). Methods fordetection of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂,include spectrophotometry, mass spectroscopy, thin layer chromatography(TLC) and reverse phase HPLC.

[0091] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0092] a) contacting succinyl-CoA and glycine with a 5-Aminolevulinatesynthase;

[0093] b) contacting succinyl-CoA and glycine with 5-Aminolevulinatesynthase and a test compound; and

[0094] c) determining the change in concentration for at least one ofthe following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/orCO₂.

[0095] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0096] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0097] a) contacting 5-aminolevulinate, CoA, and CO₂ with a5-Aminolevulinate synthase;

[0098] b) contacting 5-aminolevulinate, CoA, and CO₂ with a5-Aminolevulinate synthase and a test compound; and

[0099] c) determining the change in concentration for at least one ofthe following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/orCO₂.

[0100] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0101] Enzymatically active fragments of a fungal 5-Aminolevulinatesynthase are also useful in the methods of the invention. For example,an enzymatically active polypeptide comprising at least 100 consecutiveamino acid residues of a fungal 5-Aminolevulinate synthase may be usedin the methods of the invention. In addition, an enzymatically activepolypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98%sequence identity with a fungal 5-Aminolevulinate synthase may be usedin the methods of the invention. Most preferably, the polypeptide has atleast 50% sequence identity with a fungal 5-Aminolevulinate synthase andat least 10%, 25%, 75% or at least 90% of the activity thereof.

[0102] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0103] a) contacting succinyl-CoA and glycine with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a 5-Aminolevulinate synthase; a polypeptidehaving at least 50% sequence identity with a 5-Aminolevulinate synthaseand having at least 10% of the activity thereof;

[0104] and a polypeptide comprising at least 100 consecutive amino acidsof a 5-Aminolevulinate synthase;

[0105] b) contacting succinyl-CoA and glycine with said polypeptide anda test compound; and

[0106] c) determining the change in concentration for at least one ofthe following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/orCO₂;

[0107] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0108] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0109] a) contacting 5-aminolevulinate, CoA, and CO₂ with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a 5-Aminolevulinate synthase; a polypeptidehaving at least 50% sequence identity with a 5-Aminolevulinate synthaseand at least 10% of the activity thereof; and a polypeptide comprisingat least 100 consecutive amino acids of a 5-Aminolevulinate synthase;

[0110] b) contacting 5-aminolevulinate, CoA, and CO₂, with saidpolypeptide and a test compound; and

[0111] c) determining the change in concentration for at least one ofthe following, succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/orCO₂;

[0112] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0113] For the in vitro enzymatic assays, 5-Aminolevulinate synthaseprotein and derivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of 5-Aminolevulinate synthase maybe described in Volland and Felix (1984) Eur J Biochem 142: 551-7 (PMID:6381051). Other methods for the purification of 5-Aminolevulinatesynthase proteins and polypeptides are known to those skilled in theart.

[0114] As an alternative to in vitro assays, the invention also providescell based assays. In one embodiment, the invention provides a methodfor identifying a test compound as a candidate for an antibiotic,comprising:

[0115] a) measuring the expression of a 5-Aminolevulinate synthase in acell, cells, tissue, or an organism in the absence of a test compound;

[0116] b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of said 5-Aminolevulinatesynthase in said cell, cells, tissue, or organism; and

[0117] c) comparing the expression of 5-Aminolevulinate synthase insteps (a) and (b);

[0118] wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.

[0119] Expression of 5-Aminolevulinate synthase can be measured bydetecting the ALAS1 primary transcript or mRNA, 5-Aminolevulinatesynthase polypeptide, or 5-Aminolevulinate synthase enzymatic activity.Methods for detecting the expression of RNA and proteins are known tothose skilled in the art. See, for example, Current Protocols inMolecular Biology Ausubel et al., eds., Greene Publishing andWiley-Interscience, New York, 1995. The method of detection is notcritical to the invention. Methods for detecting ALAS1 RNA include, butare not limited to amplification assays such as quantitative reversetranscriptase-PCR, and/or hybridization assays such as Northernanalysis, dot blots, slot blots, in-situ hybridization, transcriptionalfusions using an ALAS1 promoter fused to a reporter gene, DNA assays,and microarray assays.

[0120] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect ALAS1protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with ALAS1,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

[0121] Chemicals, compounds or compositions identified by the abovemethods as modulators, preferably inhibitors, of ALAS1 expression oractivity can then be used to control fungal growth. Diseases such asrusts, mildews, and blights spread rapidly once established. Fungicidesare thus routinely applied to growing and stored crops as a preventivemeasure, generally as foliar sprays or seed dressings. For example,compounds that inhibit fungal growth can be applied to a fungus orexpressed in a fungus, in order to prevent fungal growth. Thus, theinvention provides a method for inhibiting fungal growth, comprisingcontacting a fungus with a compound identified by the methods of theinvention as having antifungal activity.

[0122] Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

[0123] Examples of undesired fungi include, but are not limited toPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

[0124] Also provided is a method of screening for an antibiotic bydetermining whether a test compound is active against the geneidentified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO:3), or the biochemical pathway or pathways it functions on.

[0125] In one particular embodiment, the method is performed byproviding an organism having a first form of the gene corresponding toeither SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutantform, a homologue, or a heterologous ALAS1 gene that performs a similarfunction as ALAS1. The first form of ALAS1 may or may not confer agrowth conditional phenotype, i.e., a 5-aminolevulinate requiringphenotype, and/or a hypersensitivity or hyposensitivity phenotype on theorganism having that altered form. In one particular embodiment a mutantform contains a transposon insertion. A comparison organism having asecond form of an ALAS1, different from the first form of the gene isalso provided, and the two organisms are separately contacted with atest compound. The growth of the two organisms in the presence of thetest compound is then compared.

[0126] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0127] a) providing cells having one form of a 5-Aminolevulinatesynthase gene, and providing comparison cells having a different form ofa 5-Aminolevulinate synthase gene; and

[0128] b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and said comparisoncells in the presence of the test compound,

[0129] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0130] It is recognized in the art that the optional determination ofthe growth of said first organism and said comparison second organism inthe absence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of an ALAS1gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

[0131] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry. New York, Worth Publishers).

[0132] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which ALAS1 functions, comprising:

[0133] a) providing cells having one form of a gene in the hemebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene;

[0134] b) contacting said cells and said comparison cells with a testcompound; and

[0135] c) determining the growth of said cells and said comparison cellsin the presence of said test compound;

[0136] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0137] The use of multi-well plates for screening is a format thatreadily accommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

[0138] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger, A., D. Nelson, etal. (1993) Principles of Biochemistry. New York, Worth Publishers).

[0139] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which ALAS1 functions, comprising:

[0140] (a) providing paired growth media comprising a first medium and asecond medium, wherein said second medium contains a higher level of5-aminolevulinate than said first medium;

[0141] (b) contacting an organism with a test compound;

[0142] (c) inoculating said first and said second media with saidorganism; and

[0143] (d) determining the growth of said organism;

[0144] wherein a difference in growth of the organism between said firstand said second media indicates that said test compound is a candidatefor an antibiotic.

[0145] It is recognized in the art that determination of the growth ofsaid organism in the paired media in the absence of any test compoundsmay be performed to control for any inherent differences in growth as aresult of the different media. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment, theorganism is Magnaporthe grisea.

EXPERIMENTAL Example 1

[0146] Construction of Plasmids with a Transposon Containing aSelectable Marker.

[0147] Construction of Sif transposon: Sif was constructed using theGPS3 vector from the GPS-M mutagenesis system from New England Biolabs,Inc. (Beverly, Mass.) as a backbone. This system is based on thebacterial transposon Tn7. The following manipulations were done to GPS3according to Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. The kanamycin resistancegene (npt) contained between the Tn7 arms was removed by EcoRVdigestion. The bacterial hygromycin B phosphotransferase (hph) gene(Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) under controlof the Aspergillus nidulans trpc promoter and terminator (Mullaney etal. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158796)) was cloned by aHpaI/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yieldingpSif1. Excision of the ampicillin resistance gene (bla) from pSif1 wasachieved by cutting pSif1 with XmnI and Bg1I followed by a T4 DNApolymerase treatment to remove the 3′ overhangs left by the BglIdigestion and religation of the plasmid to yield pSif. Top 10F′electrocompetent E. coli cells (Invitrogen) were transformed withligation mixture according to manufacturer's recommendations.Transformants containing the Sif transposon were selected on LB agar(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press.) containing 50 ug/ml of hygromycin B(Sigma Chem. Co., St. Louis, Mo.).

Example 2

[0148] Construction of a Fungal Cosmid Library

[0149] Cosmid libraries were constructed in the pcosKA5 vector (Hamer etal (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) asdescribed in Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. Cosmid libraries werequality checked by pulsed-field gel electrophoresis, restrictiondigestion analysis, and PCR identification of single genes.

Example 3

[0150] Construction of Cosmids with Transposon Insertion into FungalGenes

[0151] Sif Transposition into a Cosmid: Transposition of Sif into thecosmid framework was carried out as described by the GPS-M mutagenesissystem (New England Biolabs, Inc.). Briefly, 2 ul of the 10×GPS buffer,70 ng of supercoiled pSIF, 8-12 μg of target cosmid DNA were mixed andtaken to a final volume of 20 ul with water. 1 ul of transposase(TnsABC) was added to the reaction and incubated for 10 minutes at 37°C. to allow the assembly reaction to happen. After the assembly reaction1 ul of start solution was added to the tube, mixed well and incubatedfor 1 hour at 37° C. followed by heat inactivation of the proteins at75° C. for 10 min. Destruction of the remaining untransposed pSif wasdone by PISceI digestion at 37° C. for 2 hours followed by 10 minincubation at 75° C. to inactivate the proteins. Transformation of Top10F′ electrocompetent cells (Invitrogen) was done according tomanufacturers recommendations. Sif-containing cosmid transformants wereselected by growth on LB agar plates containing 50 ug/ml of hygromycin B(Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

Example 4

[0152] High Throughput Preparation and Verification of TransposonInsertion into the M. grisea ALAS1 Gene

[0153]E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37 C overnight. E.coli cells were pelleted by centrifugation and cosmids were isolated bya modified alkaline lysis method (Marra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

[0154] DNA sequences adjacent to the site of the insertion werecollected and used to search DNA and protein databases using the BLASTalgorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402(PMID: 9254694)). A single insertion of SIF into the Magnaporthe griseaALAS1 gene was chosen for further analysis. This construct wasdesignated cpgmra0011005e01 and it contains the SIF transposon atapproximately amino acid 100 relative to the Aspergillus nidulanshomologue HEMA (total length: 648 amino acids, GENBANK: 585244(SWISS-PROT: P38092)).

Example 5

[0155] Preparation of ALAS1 Cosmid DNA and Transformation of Magnaporthegrisea

[0156] Cosmid DNA from the ALAS1 transposon tagged cosmid clone wasprepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI(New England Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50ul protoplast suspension was mixed with 10-20 ugof the cosmid DNA and pulsed using Gene Pulser II (BioRad) set with thefollowing parameters: resistance 200 ohm, capacitance 25 uF, voltage 0.6kV. Transformed protoplasts were regenerated in complete agar media (CM,Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) with theaddition of 20% sucrose and 200 μM 5-aminolevulinic acid (Sigma-AldritchCo.) for one day, then overlayed with CM agar media containinghygromycin B (250 ug/ml) to select transformants. Transfornants werescreened for homologous recombination events in the target gene by PCR(Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID:11296265)). Two independent strains were identified and are herebyreferred to as KO1-1 and KO1-106, respectively.

Example 6

[0157] Effect of Transposon Insertion on Magnaporthe Pathogenicity

[0158] The target fungal strains, KO1-1 and KO1-106, obtained in Example5 and the wild type strain, Guy11, were subjected to a pathogenicityassay to observe infection over a 1-week period. Rice infection assayswere performed using Indian rice cultivar CO39 essentially as describedin Valent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). Allthree strains were grown for spore production on complete agar media.Spores were harvested and the concentration of spores adjusted for wholeplant inoculations. Two-week-old seedlings of cultivar CO39 were sprayedwith 12 ml of conidial suspension (5×10⁴ conidia per ml in 0.01%Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The inoculatedplants were incubated in a dew chamber at 27° C. in the dark for 36hours, and transferred to a growth chamber (27° C. 12 hours/21° C. 12hours 70% humidity) for an additional 5.5 days. Leaf samples were takenat 3, 5, and 7 days post-inoculation and examined for signs ofsuccessful infection (i.e. lesions). FIG. 2 shows the effects of ALAS1gene disruption on Magnaporthe infection at five days post-inoculation.

Example 7

[0159] Verification of ALAS1 Gene Function by Analysis of NutritionalRequirements

[0160] The fungal strains, KO1-1 and KO1-106, containing the ALAS1disrupted gene obtained in Example 5 were analyzed for their nutritionalrequirement for 5-aminolevulinic acid using the PM5 phenotype microarrayfrom Biolog, Inc. (Hayward, Calif.). The PM5 plate tests for theauxotrophic requirement for 94 different metabolites. The inoculatingfluid consists of 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5mM NaNO₃, 6.7 mM KCl, 3.5 mM Na₂SO₄, 11 mM KH₂PO₄, 0.01%p-iodonitrotetrazolium violet, 0.1 mM MgCl₂, 1.0 mM CaCl₂ and traceelements. Final concentrations of trace elements are: 7.6 μM ZnCl₂, 2.5μM MnCl₂4H₂O, 1.8 μM FeCl₂ 4H₂O, 0.71 μM CoCl₂, 6H₂O, 0.64 μM CuCl₂2H₂O, 0.62 μM Na₂MoO₄, 18 μM H₃BO₃. pH adjusted to 6.0 with NaOH. Sporesfor each strain were harvested into the inoculating fluid. The sporeconcentrations were adjusted to 2×10⁵ spores/ml. 100 μl of sporesuspension were deposited into each well of the microtiter plates. Theplates were incubated at 25° C. for 7 days. Optical density (OD)measurements at 490 nm and 750 nm were taken daily. The OD₄₉₀ measuresthe extent of tetrazolium dye reduction and the level of growth, andOD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Data confirming theannotated gene function is presented in FIG. 3, and as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of5-aminolevulinate.

Example 8

[0161] Cloning and Expression Strategies, Extraction and Purification of5-Aminolevulinate Synthase Protein.

[0162] The following protocol may be employed to obtain a purified5-Aminolevulinate synthase protein.

[0163] Cloning and expression strategies:

[0164] An ALAS1 cDNA gene can be cloned into E. coli (pETvectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen)expression vectors containing His/fusion protein tags, and theexpression of recombinant protein can be evaluated by SDS-PAGE andWestern blot analysis.

[0165] Extraction:

[0166] Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer by sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

[0167] Purification:

[0168] Purify recombinant protein by Ni-NTA affinity chromatography(Qiagen).

[0169] Purification protocol: perform all steps at 4° C.:

[0170] Use 3 ml Ni-beads (Qiagen)

[0171] Equilibrate column with the buffer

[0172] Load protein extract

[0173] Wash with the equilibration buffer

[0174] Elute bound protein with 0.5 M imidazole

Example 9

[0175] Assays for Testing Binding of Test Compounds to 5-AminolevulinateSynthase

[0176] The following protocol may be employed to identify test compoundsthat bind to the 5-Aminolevulinate synthase protein.

[0177] Purified full-length 5-Aminolevulinate synthase polypeptide witha His/fusion protein tag (Example 8) is bound to a HisGrab™ NickelCoated Plate (Pierce, Rockford, Ill.) following manufacturer'sinstructions.

[0178] Buffer conditions are optimized (e.g. ionic strength or pH,Shoolingin-Jordan et al. (1997) Methods Enzymol 281: 309-16 (PMID:9250995)) for binding of radiolabeled succinyl-CoA (custom made,PerkinElmer Life Sciences, Inc., Boston, Mass.) to the bound5-Aminolevulinate synthase.

[0179] Screening of test compounds is performed by adding test compoundand radiolabeled succinyl-CoA (custom made, PerkinElmer Life Sciences,Inc., Boston, Mass.) to the wells of the HisGrab™ plate containing bound5-Aminolevulinate synthase.

[0180] The wells are washed to remove excess labeled ligand andscintillation fluid (Scintiverse®, Fisher Scientific) is added to eachwell.

[0181] The plates are read in a microplate scintillation counter.

[0182] Candidate compounds are identified as wells with lowerradioactivity as compared to control wells with no test compound added.

[0183] Additionally, a purified polypeptide comprising 10-50 amino acidsfrom the M. grisea 5-Aminolevulinate synthase is screened in the sameway. A polypeptide comprising 10-50 amino acids is generated bysubcloning a portion of the ALAS1 gene into a protein expression vectorthat adds a His-Tag when expressed (see Example 8). Oligonucleotideprimers are designed to amplify a portion of the ALAS1 gene using thepolymerase chain reaction amplification method. The DNA fragmentencoding a polypeptide of 10-50 amino acids is cloned into an expressionvector, expressed in a host organism and purified as described inExample 8 above.

[0184] Test compounds that bind ALAS1 are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al. (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

Example 10

[0185] Assays for Testing Inhibitors or Candidates for Inhibition of5-Aminolevulinate Synthase Activity

[0186] The enzymatic activity of 5-Aminolevulinate synthase isdetermined in the presence and absence of candidate compounds in asuitable reaction mixture, such as described by Shoolingin-Jordan et al.(1997) Methods Enzymol 281: 309-16 (PMID: 9250995). Candidate compoundsare identified when a decrease in products or a lack of decrease insubstrates is detected with the reaction proceeding in either direction.

[0187] Additionally, the enzymatic activity of a polypeptide comprising10-50 amino acids from the M. grisea 5-Aminolevulinate synthase isdetermined in the presence and absence of candidate compounds in asuitable reaction mixture, such as described by Shoolingin-Jordan et al.(1997) Methods Enzymol 281: 309-16 (PMID: 9250995). A polypeptidecomprising 10-50 amino acids is generated by subdloning a portion of theALAS1 gene into a protein expression vector that adds a His-Tag whenexpressed (see Example 8). Oligonucleotide primers are designed toamplify a portion of the ALAS1 gene using polymerase chain reactionamplification method. The DNA fragment encoding a polypeptide of 10-50amino acids is cloned into an expression vector, expressed and purifiedas described in Example 8 above.

[0188] Test compounds identified as inhibitors of ALAS1 activity arefurther tested for antibiotic activity. Magnaporthe grisea fungal cellsare grown under standard fungal growth conditions that are well knownand described in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et al. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

Example 11

[0189] Assays for Testing Compounds for Alteration of 5-AminolevulinateSynthase Gene Expression

[0190]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on completeagar or oatmeal agar media after growth for 10-13 days in the light at25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml. 25 mlcultures are prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the ALAS1 gene as a probe. Testcompounds resulting in a reduced level of ALAS1 MRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

Example 12

[0191] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of 5-Aminolevulinate Synthase with No Activity

[0192]Magnaporthe grisea fungal cells containing a mutant form of theALAS1 gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 200 μM 5-aminolevulinate (Sigma-Aldrich Co.)after growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium containing 20 μM 5-aminolevulinate to a concentration of 2×10⁵spores per ml. Approximately 4×10⁴ spores are added to each well of96-well plates to which a test compound is added (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present (growth control), and wells without cells areincluded as controls (negative control). The plates are incubated at 25°C. for seven days and optical density measurements at 590 nm are takendaily. Wild type cells are screened under the same conditions. Theeffect of each compound on the mutant and wild-type fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26:177-221 (PMID: 7749303)).

Example 13

[0193] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of 5-Aminolevulinate Synthase with ReducedActivity

[0194]Magnaporthe grisea fungal cells containing a mutant form of theALAS1 gene, such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 200μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10-13 days inthe light at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores are added to each well of 96-wellplates to which a test compound is added (at varying concentrations).The total volume in each well is 200 μl. Wells with no test compoundpresent (growth control), and wells without cells are included ascontrols (negative control). The plates are incubated at 25° C. forseven days and optical density measurements at 590 nm are taken daily.Wild type cells are screened under the same conditions. The effect ofeach compound on the mutant and wild-type fungal strains is measuredagainst the growth control and the percent of inhibition is calculatedas the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild-type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 14

[0195] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of a Heme Biosynthetic Gene with No Activity

[0196]Magnaporthe grisea fungal cells containing a mutant form of a genein the heme biosynthetic pathway (e.g. Aminolevulinate dehydratase (E.C.4.2.1.24)) are grown under standard fungal growth conditions that arewell known and described in the art. Magnaporthe grisea spores areharvested from cultures grown on complete agar medium containing 200 μM5-aminolevulinate (Sigma-Aldrich Co.) after growth for 10-13 days in thelight at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium containing 20 μM 5-aminolevulinateto a concentration of2×10⁵ spores per ml. Approximately 4×10⁴ spores orcells are harvested and added to each well of 96-well plates to whichgrowth media is added in addition to an amount of test compound (atvarying concentrations). The total volume in each well is 200 μl. Wellswith no test compound present, and wells without cells are included ascontrols. The plates are incubated at 25° C. for seven days and opticaldensity measurements at 590 nm are taken daily. Wild type cells arescreened under the same conditions. The effect of each compound on themutant and wild-type fungal strains is measured against the growthcontrol and the percent of inhibition is calculated as the OD₅₉₀ (fungalstrain plus test compound)/OD₅₉₀ (growth control)×100. The percent ofgrowth inhibition as a result of a test compound on a fungal strain andthat on the wild type cells are compared. Compounds that showdifferential growth inhibition between the mutant and the wild-type areidentified as potential antifungal compounds. Similar protocols may befound in Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:7749303)).

Example 15

[0197] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining a Mutant Form of a Heme Biosynthetic Gene with ReducedActivity

[0198]Magnaporthe grisea fungal cells containing a mutant form of a genein the heme biosynthetic pathway (e.g. Aminolevulinate dehydratase (E.C.4.2.1.24)), such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungalcells containing a mutant form of are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 200 μM 5-aminolevulinate (Sigma-Aldrich Co.) after growth for10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 16

[0199] In Vivo Cell Based Assay Screening Protocol with a Fungal StrainContaining M. grisea ALAS1 and a Second Fungal Strain Containing aHeterologous ALAS1 Gene

[0200] Wild-type Magnaporthe grisea fungal cells and M. grisea fungalcells lacking a functional ALAS1 gene and containing a 5-AminolevulinicAcid Synthase gene from Candida albicans (Genbank: 10720014, SWISS-PROT:O94069, 54% sequence identity) are grown under standard fungal growthconditions that are well known and described in the art. A M. griseastrain carrying a heterologous ALAS1 gene is made as follows:

[0201] A M. grisea strain is made with a nonfunctional ALAS1 gene, suchas one containing a transposon insertion in the native gene (seeExamples 4 and 5).

[0202] A construct containing a heterologous ALAS1 gene is made bycloning the 5-Aminolevulinic Acid Synthase gene from Candida albicansinto a fungal expression vector containing a trpC promoter andterminator (e.g. pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41:22) using standard molecular biology techniques that are well known anddescribed in the art (Sambrook et al. (1989) Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratory Press).

[0203] The said construct is used to transform the M. grisea strainlacking a functional ALAS1 gene (see Example 5). Transformants areselected on minimal agar medium lacking 5-aminolevulinate. Onlytransformants carrying a functional ALAS1 gene will grow.

[0204] Wild-type strains of Magnaporthe grisea and strains containing aheterologous form of ALAS1 are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumafter growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores or cells are harvested and added to each well of 96-well platesto which growth media is added in addition to an amount of test compound(at varying concentrations). The total volume in each well is 200 μl.Wells with no test compound present, and wells without cells areincluded as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The effectof each compound on the wild-type and heterologous fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on the wild-type and heterologous fungal strains are compared.Compounds that show differential growth inhibition between the wild-typeand heterologous strains are identified as potential antifungalcompounds with specificity to the native or heterologous ALAS1 geneproducts. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

Example 17

[0205] Pathway Specific In Vivo Assay Screening Protocol

[0206]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on oatmealagar media after growth for 10-13 days in the light at 25° C. using amoistened cotton swab. The concentration of spores is determined using ahemocytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 200 μM 5-aminolevulinate(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 200 μM 5-aminolevulinate. Test compounds are added towells containing spores in minimal media and minimal media containing5-aminolevulinate. The total volume in each well is 200 μl. Both minimalmedia and 5-aminolevulinate containing media wells with no test compoundare provided as controls. The plates are incubated at 25° C. for sevendays and optical density measurements at 590 nm are taken daily. Acompound is identified as a candidate for an antibiotic acting againstthe heme biosynthetic pathway when the observed growth in the wellcontaining minimal media is less than the observed growth in the wellcontaining 5-aminolevulinate as a result of the addition of the testcompound. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

[0207] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may be made and still fall within the scopeof the invention. The foregoing examples are intended to exemplifyvarious specific embodiments of the invention and do not limit its scopein any manner.

1 3 1 1848 DNA Magnaporthe grisea 1 atggacgccg tacttcgtca gtccaaggccttgtgcccct tcctcaagaa ggcgtcgccg 60 gccactctgc ggcagctctc gacagcatctgcaccggcca ggccccgtgt gtcaccatgt 120 ggtggtacca tctccaagct gcagctgctggcgcaccggt gccccgtcat gggccaggca 180 atggctgtcc agtcggctaa ggcgggtacgaagctgcccg tcggcctggc caagatacac 240 acttcgcgga gccaggatgc tagggccgttgacgggcctg tcgttgcttc tcgcgagaac 300 gtgcccttcc cgcccaaggc tgcttccggccgatccgccg cctcgtcgaa ccccgccgcg 360 aacgcctccc cggccgccgg ccacccgggcaagaagtttt cctatgagcg attctacgaa 420 tccgaactgc agaagaagca caaggacaaatcgtaccgct acttcaacaa catcaaccgc 480 ctggccaagg agttcccccg cgctcacatgtcggacaagg aggacaaggt gaccgtttgg 540 tgcgccaacg actacctggg catgggccgcaaccccaggg tcttgtccaa gatgcacgag 600 acgctcgatg agtacggtgc tggcgccggtggtactcgca acatctctgg ccacaaccgc 660 cacgctgtcg agctcgaggg taccatcgccaagttgcacg ccaaggattc tgcgctggtt 720 ttcagctcct gctatgttgc caacgatgccacactcgcca ctctgggtag caaaatgccg 780 gattgtgtga tcctgtcgga cagcctaaaccatgcttcca tgatccaggg tatccgccat 840 tccggcacca agaaaatcgt cttcaagcacaacgacgtag cggacctcga ggccaagctg 900 gcatccctgc cgctacatgt gcccaagatcatcgcctttg agtcagttta cagcatgtgc 960 ggctccatcg gccccattga ggagatttgcgatttggccg acaagtatgg tgctatcacc 1020 ttcctcgacg aagttcacgc cgttggtatgtacggcccac acggagccgg cgtggctgag 1080 cacctcgact ttgaggccca taaggccggccggccccgcg gcaccatcat ggaccgtatc 1140 gatattatca ccggaacgct tggcaaagcttacggctgcg ttggcggcta catcgccggt 1200 tcggccaagc tcatcgacat gatccgctcgctcgcgccag gcttcatctt caccacctct 1260 cttccgcctg cgaccatggc cggtgctcgcgccgccattg aataccagat ggagcacgac 1320 ggcgaccgca ggctgcagca gctgcacacgcgcgccgtca aggaggctct tcaacatcga 1380 gatatccccg tcatcccgaa cccgtctcacatcatcccga tcctcgttgg caacgccgag 1440 ctcgcaaagc gcgcctcgga catgctgctgtctgactacc agatctacgt acagtccatc 1500 aactacccga ccgtgcccgt cggccaggagagactgcgcg tcacccccac tcccggccac 1560 gtcaaggagt tccgtgacga cctcgtcgttgccgtcgacg ctatctggac caagctcggc 1620 atcaagcgca cctcagagtg ggctgccgagggcggcttca tcggtgtcgg cgaggagggt 1680 tccgaggccc aggcgcagcc gctgtggaccgatgcccaac tcggcatcga gcaggccgcc 1740 aaggagatca tggccttggg cactgccccgaccggctgct tcaccgagtc gcttatcgag 1800 cgcgagggcg cggctctggg ccgcggcagcatggcggctg ctgcctaa 1848 2 2275 DNA Magnaporthe grisea 2 ccagtcaaaccgaggttttg aaacggaaca agaagaaaac agaaaaagaa acccaaacaa 60 gagaagaagaaaaagaaaga agaagaaaac caatcatgga cgccgtactt cgtcagtcca 120 aggccttgtgccccttcctc aagaaggcgt cgccggccac tctgcggcag ctctcgacag 180 catctgcaccggccaggccc cgtgtgtcac catgtggtgg taccatctcc aagctgcagc 240 tgctggcgcaccggtgcccc gtcatgggcc aggcaatggc tgtccagtcg gctaaggcgg 300 gtacgaagctgcccgtcggc ctggccaaga tacacacttc gcggagccag gatgctaggg 360 ccgttgacgggcctgtcgtt gcttctcgcg agaacggtaa gtagccagcc aattggttca 420 atccccttcatccctttgca ttcgcatagt tgcaaaccgg cgctgacgtg cttccctttg 480 agaatagtgcccttcccgcc caaggctgct tccggccgat ccgccgcctc gtcgaacccc 540 gccgcgaacgcctccccggc cgccggccac ccgggcaaga agttttccta tgagcgattc 600 tacgaatccgaactgcagaa gaagcacaag gacaaatcgt accgctactt caacaacatc 660 aaccgcctggccaaggagtt cccccgcgct cacatgtcgg acaaggagga caaggtgacc 720 gtttggtgcgccaacgacta cctgggcatg ggccgcaacc ccagggtctt gtccaagatg 780 cacgagacgctcgatgagta cggtgctggc gccggtggta ctcgcaacat ctctggccac 840 aaccgccacgctgtcgagct cgagggtacc atcgccaagt tgcacgccaa ggattctgcg 900 ctggttttcagctcctgcta tgttgccaac gatgccacac tcgccactct gggtagcaaa 960 atgccggattgtgtgatcct gtcggacagc ctaaaccatg cttccatgat ccagggtatc 1020 cgccattccggcaccaagaa aatcgtcttc aagcacaacg acgtagcgga cctcgaggcc 1080 aagctggcatccctgccgct acatgtgccc aagatcatcg cctttgagtc agtttacagc 1140 atgtgcggctccatcggccc cattgaggag atttgcgatt tggccgacaa gtatggtgct 1200 atcaccttcctcgacgaagt tcacgccgtt ggtatgtacg gcccacacgg agccggcgtg 1260 gctgagcacctcgactttga ggcccataag gccggccggc cccgcggcac catcatggac 1320 cgtatcgatattatcaccgg aacgcttggc aaagcttacg gctgcgttgg cggctacatc 1380 gccggttcggccaagctcat cgacatgatc cgctcgctcg cgccaggctt catcttcacc 1440 acctctcttccgcctgcgac catggccggt gctcgcgccg ccattgaata ccagatggag 1500 cacgacggcgaccgcaggct gcagcagctg cacacgcgcg ccgtcaagga ggctcttcaa 1560 catcgagatatccccgtcat cccgaacccg tctcacatca tcccgatcct cgttggcaac 1620 gccgagctcgcaaagcgcgc ctcggacatg ctgctgtctg actaccagat ctacgtacag 1680 tccatcaactacccgaccgt gcccgtcggc caggagagac tgcgcgtcac ccccactccc 1740 ggccacgtcaaggagttccg tgacgacctc gtcgttgccg tcgacgctat ctggaccaag 1800 ctcggcatcaagcgcacctc agagtgggct gccgagggcg gcttcatcgg tgtcggcgag 1860 gagggttccgaggcccaggc gcagccgctg tggaccgatg cccaactcgg catcgagcag 1920 gccgccaaggagatcatggc cttgggcact gccccgaccg gctgcttcac cgagtcgctt 1980 atcgagcgcgagggcgcggc tctgggccgc ggcagcatgg cggctgctgc ctaagttcag 2040 ggacgtgaaatcccattgcg cgactgctat ttgatctgct tcagaatagc tatcgtttct 2100 cacctagcgacatgcaaatg tttcaatctg tggacatact acccatcgga gagttgcgca 2160 tgtagcattgcaatgttggc actatcttca caagtatata aatgatcaat gcgttagata 2220 gttgacacctagtacggata acgtacttga agagttaaac tcgattcctc ggaat 2275 3 615 PRTMagnaporthe grisea 3 Met Asp Ala Val Leu Arg Gln Ser Lys Ala Leu Cys ProPhe Leu Lys 1 5 10 15 Lys Ala Ser Pro Ala Thr Leu Arg Gln Leu Ser ThrAla Ser Ala Pro 20 25 30 Ala Arg Pro Arg Val Ser Pro Cys Gly Gly Thr IleSer Lys Leu Gln 35 40 45 Leu Leu Ala His Arg Cys Pro Val Met Gly Gln AlaMet Ala Val Gln 50 55 60 Ser Ala Lys Ala Gly Thr Lys Leu Pro Val Gly LeuAla Lys Ile His 65 70 75 80 Thr Ser Arg Ser Gln Asp Ala Arg Ala Val AspGly Pro Val Val Ala 85 90 95 Ser Arg Glu Asn Val Pro Phe Pro Pro Lys AlaAla Ser Gly Arg Ser 100 105 110 Ala Ala Ser Ser Asn Pro Ala Ala Asn AlaSer Pro Ala Ala Gly His 115 120 125 Pro Gly Lys Lys Phe Ser Tyr Glu ArgPhe Tyr Glu Ser Glu Leu Gln 130 135 140 Lys Lys His Lys Asp Lys Ser TyrArg Tyr Phe Asn Asn Ile Asn Arg 145 150 155 160 Leu Ala Lys Glu Phe ProArg Ala His Met Ser Asp Lys Glu Asp Lys 165 170 175 Val Thr Val Trp CysAla Asn Asp Tyr Leu Gly Met Gly Arg Asn Pro 180 185 190 Arg Val Leu SerLys Met His Glu Thr Leu Asp Glu Tyr Gly Ala Gly 195 200 205 Ala Gly GlyThr Arg Asn Ile Ser Gly His Asn Arg His Ala Val Glu 210 215 220 Leu GluGly Thr Ile Ala Lys Leu His Ala Lys Asp Ser Ala Leu Val 225 230 235 240Phe Ser Ser Cys Tyr Val Ala Asn Asp Ala Thr Leu Ala Thr Leu Gly 245 250255 Ser Lys Met Pro Asp Cys Val Ile Leu Ser Asp Ser Leu Asn His Ala 260265 270 Ser Met Ile Gln Gly Ile Arg His Ser Gly Thr Lys Lys Ile Val Phe275 280 285 Lys His Asn Asp Val Ala Asp Leu Glu Ala Lys Leu Ala Ser LeuPro 290 295 300 Leu His Val Pro Lys Ile Ile Ala Phe Glu Ser Val Tyr SerMet Cys 305 310 315 320 Gly Ser Ile Gly Pro Ile Glu Glu Ile Cys Asp LeuAla Asp Lys Tyr 325 330 335 Gly Ala Ile Thr Phe Leu Asp Glu Val His AlaVal Gly Met Tyr Gly 340 345 350 Pro His Gly Ala Gly Val Ala Glu His LeuAsp Phe Glu Ala His Lys 355 360 365 Ala Gly Arg Pro Arg Gly Thr Ile MetAsp Arg Ile Asp Ile Ile Thr 370 375 380 Gly Thr Leu Gly Lys Ala Tyr GlyCys Val Gly Gly Tyr Ile Ala Gly 385 390 395 400 Ser Ala Lys Leu Ile AspMet Ile Arg Ser Leu Ala Pro Gly Phe Ile 405 410 415 Phe Thr Thr Ser LeuPro Pro Ala Thr Met Ala Gly Ala Arg Ala Ala 420 425 430 Ile Glu Tyr GlnMet Glu His Asp Gly Asp Arg Arg Leu Gln Gln Leu 435 440 445 His Thr ArgAla Val Lys Glu Ala Leu Gln His Arg Asp Ile Pro Val 450 455 460 Ile ProAsn Pro Ser His Ile Ile Pro Ile Leu Val Gly Asn Ala Glu 465 470 475 480Leu Ala Lys Arg Ala Ser Asp Met Leu Leu Ser Asp Tyr Gln Ile Tyr 485 490495 Val Gln Ser Ile Asn Tyr Pro Thr Val Pro Val Gly Gln Glu Arg Leu 500505 510 Arg Val Thr Pro Thr Pro Gly His Val Lys Glu Phe Arg Asp Asp Leu515 520 525 Val Val Ala Val Asp Ala Ile Trp Thr Lys Leu Gly Ile Lys ArgThr 530 535 540 Ser Glu Trp Ala Ala Glu Gly Gly Phe Ile Gly Val Gly GluGlu Gly 545 550 555 560 Ser Glu Ala Gln Ala Gln Pro Leu Trp Thr Asp AlaGln Leu Gly Ile 565 570 575 Glu Gln Ala Ala Lys Glu Ile Met Ala Leu GlyThr Ala Pro Thr Gly 580 585 590 Cys Phe Thr Glu Ser Leu Ile Glu Arg GluGly Ala Ala Leu Gly Arg 595 600 605 Gly Ser Met Ala Ala Ala Ala 610 615

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting a5-Aminolevulinate synthase polypeptide with a test compound; and b)detecting the presence or absence of binding between said test compoundand said 5-Aminolevulinate synthase polypeptide; wherein bindingindicates that said test compound is a candidate for an antibiotic. 2.The method of claim 1, wherein said 5-Aminolevulinate synthasepolypeptide is a fungal 5-Aminolevulinate synthase polypeptide.
 3. Themethod of claim 1, wherein said 5-Aminolevulinate synthase polypeptideis a Magnaporthe 5-Aminolevulinate synthase polypeptide.
 4. The methodof claim 1, wherein said 5-Aminolevulinate synthase polypeptide is SEQID NO:
 3. 5. A method for determining whether the antibiotic candidateof claim 1 has antifungal activity, further comprising: contacting afungus or fungal cells with said antibiotic candidate and detecting thedecrease in growth, viability, or pathogenicity of said fungus or fungalcells.
 6. A method for identifying a test compound as a candidate for anantibiotic, comprising: a) contacting a test compound with at least onepolypeptide selected from the group consisting of: a polypeptide havingat least ten consecutive amino acids of a fungal 5-Aminolevulinatesynthase, a polypeptide having at least 50% sequence identity with afungal 5-Aminolevulinate synthase, and a polypeptide having at least 10%of the activity of a fungal 5-Aminolevulinate synthase; and b) detectingthe presence and/or absence of binding between said test compound andsaid polypeptide; wherein binding indicates that said test compound is acandidate for an antibiotic.
 7. A method for determining whether theantibiotic candidate of claim 6 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 8. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting succinyl-CoA and glycine with a5-Aminolevulinate synthase; b) contacting succinyl-CoA and glycine with5-Aminolevulinate synthase and a test compound; and c) determining thechange in concentration for at least one of the following: succinyl-CoA,glycine, 5-aminolevulinate, CoA, and/or CO₂; wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 9.The method of claim 8, wherein said 5-Aminolevulinate synthase is afungal 5-Aminolevulinate synthase.
 10. The method of claim 8, whereinsaid 5-Aminolevulinate synthase is a Magnaporthe 5-Aminolevulinatesynthase.
 11. The method of claim 8, wherein said 5-Aminolevulinatesynthase is SEQ ID NO:
 3. 12. A method for determining whether theantibiotic candidate of claim 8 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 13. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting 5-aminolevulinate, CoA, and CO₂ with a5-Aminolevulinate synthase; b) contacting 5-aminolevulinate, CoA, andCO₂ with a 5-Aminolevulinate synthase and a test compound; and c)determining the change in concentration for at least one of thefollowing: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂;wherein a change in concentration for any of the above substancesbetween steps (a) and (b) indicates that said test compound is acandidate for an antibiotic.
 14. The method of claim 13, wherein said5-Aminolevulinate synthase is a fungal 5-Aminolevulinate synthase. 15.The method of claim 13, wherein said 5-Aminolevulinate synthase is aMagnaporthe 5-Aminolevulinate synthase.
 16. The method of claim 13,wherein said 5-Aminolevulinate synthase is SEQ ID NO:
 3. 17. A methodfor determining whether the antibiotic candidate of claim 13 hasantifungal activity, further comprising: contacting a fungus or fungalcells with said antibiotic candidate and detecting a decrease in growth,viability, or pathogenicity of said fungus or fungal cells.
 18. A methodfor identifying a test compound as a candidate for an antibiotic,comprising: a) contacting succinyl-CoA and glycine with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with 5-Aminolevulinate synthase; a polypeptide havingat least 50% sequence identity with a 5-Aminolevulinate synthase andhaving at least 10% of the activity thereof; and a polypeptidecomprising at least 100 consecutive amino acids of a 5-Aminolevulinatesynthase; b) contacting succinyl-CoA and glycine with said polypeptideand a test compound; and c) determining the change in concentration forat least one of the following: succinyl-CoA, glycine, 5-aminolevulinate,CoA, and/or CO₂; wherein a change in concentration for any of the abovesubstances between steps (a) and (b) indicates that said test compoundis a candidate for an antibiotic.
 19. A method for identifying a testcompound as a candidate for an antibiotic, comprising: a) contacting5-aminolevulinate, CoA, and CO₂ with a polypeptide selected from thegroup consisting of: a polypeptide having at least 50% sequence identitywith a 5-Aminolevulinate synthase; a polypeptide having at least 50%sequence identity with a 5-Aminolevulinate synthase and at least 10% ofthe activity thereof; and a polypeptide comprising at least 100consecutive amino acids of a 5-Aminolevulinate synthase; b) contacting5-aminolevulinate, CoA, and CO₂, with said polypeptide and a testcompound; and c) determining the change in concentration for at leastone of the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA,and/or CO₂; wherein a change in concentration for any of the abovesubstances between steps (a) and (b) indicates that said test compoundis a candidate for an antibiotic.
 20. A method for identifying a testcompound as a candidate for an antibiotic, comprising: a) measuring theexpression of a 5-Aminolevulinate synthase in a cell, cells, tissue, oran organism in the absence of a test compound; b) contacting said cell,cells, tissue, or organism with said test compound and measuring theexpression of said 5-Aminolevulinate synthase in said cell, cells,tissue, or organism; and c) comparing the expression of5-Aminolevulinate synthase in steps (a) and (b); wherein a lowerexpression in the presence of said test compound indicates that saidtest compound is a candidate for an antibiotic.
 21. The method of claim20 wherein said cell, cells, tissue, or organism is, or is derived froma fungus.
 22. The method of claim 20 wherein said cell, cells, tissue,or organism is, or is derived from a Magnaporthe fungus or fungal cell.23. The method of claim 20, wherein said 5-Aminolevulinate synthase isSEQ ID NO:
 3. 24. The method of claim 20, wherein the expression of5-Aminolevulinate synthase is measured by detecting ALAS1 mRNA.
 25. Themethod of claim 20, wherein the expression of 5-Aminolevulinate synthaseis measured by detecting 5-Aminolevulinate synthase polypeptide.
 26. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: a) providing cells having one form of a 5-Aminolevulinatesynthase gene, and providing comparison cells having a different form ofa 5-Aminolevulinate synthase gene; and b) contacting said cells and saidcomparison cells with a test compound and determining the growth of saidcells and comparison cells in the presence of the test compound; whereina difference in growth between said cells and said comparison cells inthe presence of said compound indicates that said compound is acandidate for an antibiotic.
 27. The method of claim 26 wherein thecells and the comparison cells are fungal cells.
 28. The method of claim26 wherein the cells and the comparison cells are Magnaporthe cells. 29.The method of claim 26 wherein said form and said comparison form of the5-Aminolevulinate synthase are fungal 5-Aminolevulinate synthases. 30.The method of claim 26, wherein at least one of the forms is aMagnaporthe 5-Aminolevulinate synthase.
 31. The method of claim 26wherein said form and said comparison form of the 5-Aminolevulinatesynthase are non-fungal 5-Aminolevulinate synthases.
 32. The method ofclaim 26 wherein one form of the 5-Aminolevulinate synthase is a fungal5-Aminolevulinate synthase, and the other form is a non-fungal5-Aminolevulinate synthase.
 33. A method for identifying a test compoundas a candidate for an antibiotic, comprising: a) providing cells havingone form of a gene in the heme biochemical and/or genetic pathway andproviding comparison cells having a different form of said gene. b)contacting said cells and said comparison cells with a test compound, c)determining the growth of said cells and said comparison cells in thepresence of said test compound; wherein a difference in growth betweensaid cells and said comparison cells in the presence of said testcompound indicates that said test compound is a candidate for anantibiotic.
 34. The method of claim 33 wherein the cells and thecomparison cells are fungal cells.
 35. The method of claim 33 whereinthe cells and the comparison cells are Magnaporthe cells.
 36. The methodof claim 33 wherein said form and said different form of the hemebiosynthesis gene are fungal heme biosynthesis genes.
 37. The method ofclaim 33, wherein at least one form is a Magnaporthe heme biosynthesisgene.
 38. The method of claim 33 wherein said form and said differentform of the heme biosynthesis genes are non-fungal heme biosynthesisgenes.
 39. The method of claim 33 wherein one form of the hemebiosynthesis gene is a fungal heme biosynthesis gene, and the differentform is a non-fungal heme biosynthesis gene.
 40. A method fordetermining whether the antibiotic identified by the method of claim 33has antifungal activity, further comprising: contacting a fungus orfungal cells with said antibiotic candidate and detecting a decrease ingrowth, viability, or pathogenicity of said fungus or fungal cells,wherein a decrease in growth, viability, or pathogenicityof said fungusor fungal cells indicates that the antibiotic has antifungal activity.41. A method for identifying a test compound as a candidate for anantibiotic, comprising: (a) providing paired growth media; comprising afirst medium and a second medium, wherein said second medium contains ahigher level of 5-aminolevulinate than said first medium; (b) contactingan organism with a test compound; (c) inoculating said first and saidsecond media with said organism; and (d) determining the growth of saidorganism; wherein a difference in growth of the organism between saidfirst and said second media indicates that said test compound is acandidate for an antibiotic.
 42. The method of claim 41, wherein saidorganism is a fungus.
 43. The method of claim 41, wherein said organismis Magnaporthe.
 44. An isolated nucleic acid comprising a nucleotidesequence that encodes a polypeptide of SEQ ID NO:
 3. 45. The nucleicacid of claim 44 comprising the nucleotide sequence of SEQ ID NO:
 1. 46.An expression cassette comprising the nucleic acid of claim
 45. 47. Theisolated nucleic acid of claim 44 comprising a nucleotide sequence withat least 50 to at least 95% sequence identity to SEQ ID NO:
 1. 48. Apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:
 3. 49. A polypeptide comprising the amino acid sequence of SEQ IDNO: 3.